Detection of the Sanfilippo type B syndrome using radiolabelled oligosaccharides as substrates for the estimation of alpha-N-acetylglucosaminidase.
1. The following radiolabelled disaccharides were prepared from heparin and evaluated as substrates for alpha-N-acetylglucosaminidase present in cultured skin fibroblasts: O-(alpha-3-acetamido-2-deoxy-D-glucopyranosyl)-(1 leads to 4)-L-[6,3H]idose (GlcNAc-Ido), O-(alpha-2-acetamido-2-deoxy-D-glucopyranosyl)-(1 leads to 4)-1,6 anhydro-L-[6,3H]idose (GlcNAc-anIdo), O-(alpha-2-acetamido-2-deoxy-D-glucopyranosyl)-(1 leads to 4)-L-[6,3H]idose 2-sulfate (GlcNAc-Ido(OS)), O-(alpha 2-acetamido-2-deoxy-D-glucopyranosyl)-(1 leads to 3)-L-[6,3H]idonic acid (GlcNAc-IdOA). 2. Alpha-N-Acetylglucosaminidase activity assessed with GlcNAc-IdOA was 12 times higher than the values obtained using GlcNAc-Ido, GlcNAc-anIdo and GlcNAc-Ido(OS). Less than 5% of normal activity resulted when these substrates were incubated with fibroblasts from Sanfilippo B patients. These results demonstrate that a C6 carboxyl group on the adjacent residue to the N-acetylglucosaminide moiety is an important structural requirement in the mechanism of action or binding of alpha-N-acetylglucosaminidase toward alpha-linked N-acetylglucosaminide residues. The presence of a C2 sulfate group on the adjacent residue had no effect on enzyme activity. 3. Alpha-N-Acetylglucosaminidase activity in leucocyte and fibroblast homogenates assayed using GlcNAc-IdOA as substrate clearly distinguished Sanfilippo B patients from normal controls, and Sanfilippo A, C and D patients.
D000117:Acetylglucosamine / Q000378:metabolism; D000118:Acetylglucosaminidase / Q000378:metabolism*; D002478:Cells, Cultured; D004187:Disaccharides / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology; D006596:Hexosaminidases / Q000378:metabolism*; D006801:Humans; D007962:Leukocytes / Q000201:enzymology*; D009083:Mucopolysaccharidoses / Q000175:diagnosis*; D009084:Mucopolysaccharidosis III / Q000175:diagnosis* / Q000201:enzymology; D012867:Skin / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014316:Tritium
D004187:Disaccharides; D014316:Tritium; D006596:Hexosaminidases; D000118:Acetylglucosaminidase; D000117:Acetylglucosamine